In situ hybridization is a method used to localize nucleic acid sequences (either DNA or RNA) in the cytoplasm, organelles, chromosomes or nuclei of biological material. The ability to detect nuclei acids in situ allows construction of physical maps of chromosomes, analysis of chromosome structure and aberration, investigation of the structure function and evolution of chromosomes and genomes, determination of the spatial and temporal expression of genes. In situ hybridization shows chromosome rearrangement and changes in sequence abundance during evolution, organism development and disease.
DNA in situ hybridisation to chromosomal targets has become tantamount with fluorescent in situ hybridisation. In comparison to other methods, fluorescent detection of hybridization sites has advantages on resolution, contrast speed and safety as well as the advantage of simultaneous multi target localization, which is a critical advantage. This example of in situ hybridisation simply involves the addition of a probe that is used to detect and localize the presence or absence of specific DNAsequenceson chromosomes. A fluorescent tag is added to the probe (also known as a fluorescent probe) and this binds to only parts of the chromosomes, in the fixed tissues, which complementary to it. Due to the fluorescent tag the activity of the gene and be analysed this method is used allowing chromosomal painting (which we were shown in the lecture) and even chromosomal structure and function (developmental biology)
Fundamental biological questions are answered using genomic in situ hybridisation and the method is of use in medical research and diagnosis and plant breeding programmes
In Situ Hybridisation is a widely used and mature technique, with many hundreds of laboratories publishing their methods. In Situ Hybridisation was introduced in 1969 (Buongiorno- Nardelli and Amaldi 1969;) and has been primarily for the localisation of DNA sequences. Haper and Saunders 1981 reported the first successful single copy genomic in situ hybridisation experiment localizing the human insulin gene on human chromosome 1 using radioactively labelled probes. Since then In situ hybridisation has now localized either radioactively or non-radioactively, many unique or low copy sequence including genes in humans (Trask et al) in Drosophilla (Hafen et al ) and in Caenorhabdites (e.g. muscle protein genes; Albertson 1985)
Genomic in situ hybridisation.....= GISH is conducted by inserting labelled probes into tissue where they bind to specific mRNA .....NO GISH is always DNA probes from whole genomes .and then are detected and visualised using their labels. The probes detect specific sequences of nucleotides and bind to mRNA.....NO in the nucleus. These probes are labelled by substances such as fluorescents or radioactive substances so that they can be detected and visualised. The tissue to be used is prepared usually by snap freezing or cell suspension. The cells are made more permeable so the probes can enter by placing in HCl or a detergent treatment. The probes then enter the cells and move to the nucleus where they bind to mRN. After the probes have bound they are detected using the labels applied earlier. Fluorescent labels are detected directly using a fluorescent microscope whereas radioactive labels are detected indirectly by using photographic paper; the tissue is exposed to the film which is then developed.
Genomic in situ hybridisation is a valuable resource used to look at parent gnerations of polyploid. GISH and DAPI staining can be used to illustrate which part of the polyploidy genome come from which parent, a valuable tool to define the direction of evolution.
Allopolyploids are individual organisms that are polyploidy and have chromosomes of multiple origins. It is the most common type of polyploidy (Soltis et al 200). Allopolyploids are a direct result of interbreeding or hybridization between different but closely related species. The chromosomes are thus similar and pairing is not inhibited during meiosis.
Tragopogon dubius, Tragopogon pratensis and Tragopogon porrifolius were introduced to North America from Europe. They may not have come into close contact before this time and thus may not have had an opportunity to interbreed. These ancestral species had a diploid chromosome number of 12. In the allopolyploids the diploid cells contained a 2n value of 24 chromosomes. F1 progeny, T.mirius of parent T.dubius and T.porrifolius .....should be in italics was an allopolyploid with a diploid chromosome number of 24. Similarly in f1 progeny T.miscellus of parents T.dubis and T.pratensis the diploid chromosome number was 24. Doubling of chromosome numbers results in reproductive isolation from parental generation and the F1 generation will continue to reproduce via self fertilisation as is typical for polyploidy plant populations.....or cross pollination with another allopolyploids. This last sentence is poorly constructed . The hybridization and additive mechanism that result in allopolyploid individuals can result in a variation of characteristics from.....(in the) the parental progeny (Sadava et al 20008). Although both F1 progeny display negligible changes to chromosomal arrangement they show significant differences in gene expression (Soltis et al 2004). This explains why some T.mirus grows well in environments that are more sheltered from the sun whether as neither of its paternal generation possess such characteristics (Sadava et al, 2008)
Homogenisation of DNA in polyploids is very common (Pires et al.....date?). It has been found in many species but the benefit it plays in evolution is currently unclear. Homogenisation is rapid genomic rearrangement that can sometimes result is genomic downsizing, that is the loss of DNA from one parental species.
It had been thought that the allotetraploid T.mirus had not undergone homogenisation during the emergence of the new species (Pires et al.....date?). . Homogenisation takes place within the early generations (<40) but not straight after the emergence of T.mirius (Koh et al).....needs explanation, why 40 generations for example?. However Leitch et al.....date have discovered that homogenisation indeed does take place within the T.mirius species. Pires et al may have thought homogenisation did because he may have studied very early generations of T.mirius. When studying T.mirius because it is a relatively new species it is unclear how old a population one is studying may be.
During homogenisation it is often that the ribosomal DNA of parent T.dubius is often lost.....poor sentence structure. In a study of natural populations of T.mirius, 0% of populations had reduced T.porrifolius genomes whilst 71% of populations had reduced T.dubius genomes (Malinska et al).
The metaphase karyotype of T.mirius shows an additive genotype between T.dubius and T.porrifolius, the karyotype is likely to be of an early generation since homogenisation is not noticeable.
One supposed advantage of homogenisation have been the balancing of enlarging retrotransposons (Hawkins et el). Li et al have studied that up to 68% of wheat genome may be made up of retrotransposons.
References
A.R. Leitch, T. Schwarzacher, D. Jackson and I. J. Leitch (1994). IN SITU HYBRIDIZATION. London : BIOS Scientific Publisher Limited,1994.
HAFEN E, LEVINE M, GARBER RL, GEHRING WJ(1983), AN IMPROVED INSITU HYBRIDIZATION METHOD FOR THE DETECTION OF CELLULAR RNAS IN DROSOPHILA TISSUE-SECTIONS AND ITS APPLICATION FOR LOCALIZING TRANSCRIPTS OF THE HOMEOTIC ANTENNAPEDIA GENE-COMPLEX, The EMBO Journal, 2(4), 617-623
Buongiorno- Nardelli M, Amaldi F (1970), AUTORADIOGRAPHIC DETECTION OF MOLECULAR HYBRIDS BETWEEN RRNA AND DNA IN TISSUE SECTIONS, Nature, 225(5236), 946
Li W, Zhang P, Fellers JP, Friebe B, Gill BS (2004), Sequence composition, organization and evolution of the core Triticeae genome. Plant Journal. 40 (4), 500-511.
Hawkins JS, Grover CE, Wendel JF (2008), Repeated big bangs and the expanding universe: Directionality in plant genome size evolution, Plant Science, 174 (6), 557-562.
Pires JC, Lim KY, Kovarik A, Matyasek R, Boyd A, Leitch AR, Leitch IJ, Bennett MD, Soltis PS, Soltis DE (2004), Molecular cytogenetic analysis of recently evolved Tragopogan (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors, American Journal of Botany, 91(7), 1022-1035
Koh J, Soltis PS, Soltis DE (2010), Homeolog loss and expression changes in natural population of the recently and repeatedly formed allotetraploid Tragopogon mirus (Asteraceae), BMC Genomics, 11(97).
Leitch AR, Pires JC, Kovarik A, Lim KY, Sherwood AM, Matyasek R, Rocca J, Soltis DE, Soltis PS (2005), Rapid Concerted Evolution of Nuclear Ribosomal DNA in Two Tragopogon Allopolyploids of Recent and Recurrent Origin, Genetics, 169(2), 931-944
Soltis PS, soltis DE ,2000. The role of genetic and genomic changes in the succes of polyploids, Proceedings of the National Academy of Sciences, 97, 7051-7057
Soltis DE, Soltis PS, Pires JC, Kovarik A, Tate JA, Mavrodiev E, 2004, Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons, Biological Journal of the Linnean Society. 82, 485-501 Sadava, Purves, Heller, Orians, Hellis, 2008. Life: The science of Biology. 8th Edition, Sinauer Associates. Cook LM, Soltis PS, 1999, Mating systems of diploid and allotetraploid populations of Tragopogon (Asteraceae). I. Natural Populations, Heredity, 82, 237-244
Group name: The Genies
Secretary (Email): Lizzamah Akinlade (bt09379@qmul.ac.uk)
Members (Email)
Bahja Mohammed (ef08055@qmul.ac.uk)
Femi Adebayo (femidaniel@aol.com)
Nora Mnini (ef07323@qmul.ac.uk)
Yasmine Gonaoui - Badri (ef07122@qmul.ac.uk)
In situ Hybridization
In situ hybridization is a method used to localize nucleic acid sequences (either DNA or RNA) in the cytoplasm, organelles, chromosomes or nuclei of biological material. The ability to detect nuclei acids in situ allows construction of physical maps of chromosomes, analysis of chromosome structure and aberration, investigation of the structure function and evolution of chromosomes and genomes, determination of the spatial and temporal expression of genes. In situ hybridization shows chromosome rearrangement and changes in sequence abundance during evolution, organism development and disease.
DNA in situ hybridisation to chromosomal targets has become tantamount with fluorescent in situ hybridisation. In comparison to other methods, fluorescent detection of hybridization sites has advantages on resolution, contrast speed and safety as well as the advantage of simultaneous multi target localization, which is a critical advantage. This example of in situ hybridisation simply involves the addition of a probe that is used to detect and localize the presence or absence of specific DNA sequenceson chromosomes. A fluorescent tag is added to the probe (also known as a fluorescent probe) and this binds to only parts of the chromosomes, in the fixed tissues, which complementary to it. Due to the fluorescent tag the activity of the gene and be analysed this method is used allowing chromosomal painting (which we were shown in the lecture) and even chromosomal structure and function (developmental biology)
Fundamental biological questions are answered using genomic in situ hybridisation and the method is of use in medical research and diagnosis and plant breeding programmes
In Situ Hybridisation is a widely used and mature technique, with many hundreds of laboratories publishing their methods. In Situ Hybridisation was introduced in 1969 (Buongiorno- Nardelli and Amaldi 1969;) and has been primarily for the localisation of DNA sequences. Haper and Saunders 1981 reported the first successful single copy genomic in situ hybridisation experiment localizing the human insulin gene on human chromosome 1 using radioactively labelled probes. Since then In situ hybridisation has now localized either radioactively or non-radioactively, many unique or low copy sequence including genes in humans (Trask et al) in Drosophilla (Hafen et al ) and in Caenorhabdites (e.g. muscle protein genes; Albertson 1985)
Genomic in situ hybridisation.....= GISH is conducted by inserting labelled probes into tissue where they bind to specific mRNA .....NO GISH is always DNA probes from whole genomes .and then are detected and visualised using their labels. The probes detect specific sequences of nucleotides and bind to mRNA.....NO in the nucleus. These probes are labelled by substances such as fluorescents or radioactive substances so that they can be detected and visualised. The tissue to be used is prepared usually by snap freezing or cell suspension. The cells are made more permeable so the probes can enter by placing in HCl or a detergent treatment. The probes then enter the cells and move to the nucleus where they bind to mRN. After the probes have bound they are detected using the labels applied earlier. Fluorescent labels are detected directly using a fluorescent microscope whereas radioactive labels are detected indirectly by using photographic paper; the tissue is exposed to the film which is then developed.
Genomic in situ hybridisation is a valuable resource used to look at parent gnerations of polyploid. GISH and DAPI staining can be used to illustrate which part of the polyploidy genome come from which parent, a valuable tool to define the direction of evolution.
Allopolyploids are individual organisms that are polyploidy and have chromosomes of multiple origins. It is the most common type of polyploidy (Soltis et al 200). Allopolyploids are a direct result of interbreeding or hybridization between different but closely related species. The chromosomes are thus similar and pairing is not inhibited during meiosis.
Tragopogon dubius, Tragopogon pratensis and Tragopogon porrifolius were introduced to North America from Europe. They may not have come into close contact before this time and thus may not have had an opportunity to interbreed. These ancestral species had a diploid chromosome number of 12. In the allopolyploids the diploid cells contained a 2n value of 24 chromosomes. F1 progeny, T.mirius of parent T.dubius and T.porrifolius .....should be in italics was an allopolyploid with a diploid chromosome number of 24. Similarly in f1 progeny T.miscellus of parents T.dubis and T.pratensis the diploid chromosome number was 24. Doubling of chromosome numbers results in reproductive isolation from parental generation and the F1 generation will continue to reproduce via self fertilisation as is typical for polyploidy plant populations.....or cross pollination with another allopolyploids. This last sentence is poorly constructed . The hybridization and additive mechanism that result in allopolyploid individuals can result in a variation of characteristics from.....(in the) the parental progeny (Sadava et al 20008). Although both F1 progeny display negligible changes to chromosomal arrangement they show significant differences in gene expression (Soltis et al 2004). This explains why some T.mirus grows well in environments that are more sheltered from the sun whether as neither of its paternal generation possess such characteristics (Sadava et al, 2008)
Homogenisation of DNA in polyploids is very common (Pires et al.....date?). It has been found in many species but the benefit it plays in evolution is currently unclear. Homogenisation is rapid genomic rearrangement that can sometimes result is genomic downsizing, that is the loss of DNA from one parental species.
It had been thought that the allotetraploid T.mirus had not undergone homogenisation during the emergence of the new species (Pires et al.....date?). . Homogenisation takes place within the early generations (<40) but not straight after the emergence of T.mirius (Koh et al).....needs explanation, why 40 generations for example?. However Leitch et al.....date have discovered that homogenisation indeed does take place within the T.mirius species. Pires et al may have thought homogenisation did because he may have studied very early generations of T.mirius. When studying T.mirius because it is a relatively new species it is unclear how old a population one is studying may be.
During homogenisation it is often that the ribosomal DNA of parent T.dubius is often lost.....poor sentence structure. In a study of natural populations of T.mirius, 0% of populations had reduced T.porrifolius genomes whilst 71% of populations had reduced T.dubius genomes (Malinska et al).
The metaphase karyotype of T.mirius shows an additive genotype between T.dubius and T.porrifolius, the karyotype is likely to be of an early generation since homogenisation is not noticeable.
One supposed advantage of homogenisation have been the balancing of enlarging retrotransposons (Hawkins et el). Li et al have studied that up to 68% of wheat genome may be made up of retrotransposons.
References
A.R. Leitch, T. Schwarzacher, D. Jackson and I. J. Leitch (1994). IN SITU HYBRIDIZATION. London : BIOS Scientific Publisher Limited,1994.W.A.BICKMORE (1999). Chromosome Structural Analysis . New York: Oxford Univerity Press. 1-197.
HAFEN E, LEVINE M, GARBER RL, GEHRING WJ(1983), AN IMPROVED INSITU HYBRIDIZATION METHOD FOR THE DETECTION OF CELLULAR RNAS IN DROSOPHILA TISSUE-SECTIONS AND ITS APPLICATION FOR LOCALIZING TRANSCRIPTS OF THE HOMEOTIC ANTENNAPEDIA GENE-COMPLEX, The EMBO Journal, 2(4), 617-623
Buongiorno- Nardelli M, Amaldi F (1970), AUTORADIOGRAPHIC DETECTION OF MOLECULAR HYBRIDS BETWEEN RRNA AND DNA IN TISSUE SECTIONS, Nature, 225(5236), 946
HARPER ME, SAUNDERS GF (1980), LOCALIZATION OF CLONED SINGLE COPY GENES ON HUMAN-CHROMOSOMES BY INSITU HYBRIDIZATION, European Journal of cell biology, 22(1), 20
TRASK BJ, ALLEN S, MASSA H, FERTITTA A, SACHS R, VANDENENGH G, WU M(1993), STUDIES OF METAPHASE AND INTERPHASE CHROMOSOMES USING FLUORESCENCE IN-SITU HYBRIDIZATION, Cold Spring Harbor Symposia on quantitative biology, 58, 767-775,
Li W, Zhang P, Fellers JP, Friebe B, Gill BS (2004), Sequence composition, organization and evolution of the core Triticeae genome. Plant Journal. 40 (4), 500-511.
Hawkins JS, Grover CE, Wendel JF (2008), Repeated big bangs and the expanding universe: Directionality in plant genome size evolution, Plant Science, 174 (6), 557-562.
Pires JC, Lim KY, Kovarik A, Matyasek R, Boyd A, Leitch AR, Leitch IJ, Bennett MD, Soltis PS, Soltis DE (2004), Molecular cytogenetic analysis of recently evolved Tragopogan (Asteraceae) allopolyploids reveal a karyotype that is additive of the diploid progenitors, American Journal of Botany, 91(7), 1022-1035
Koh J, Soltis PS, Soltis DE (2010), Homeolog loss and expression changes in natural population of the recently and repeatedly formed allotetraploid Tragopogon mirus (Asteraceae), BMC Genomics, 11(97).
Leitch AR, Pires JC, Kovarik A, Lim KY, Sherwood AM, Matyasek R, Rocca J, Soltis DE, Soltis PS (2005), Rapid Concerted Evolution of Nuclear Ribosomal DNA in Two Tragopogon Allopolyploids of Recent and Recurrent Origin, Genetics, 169(2), 931-944
Soltis PS, soltis DE ,2000. The role of genetic and genomic changes in the succes of polyploids, Proceedings of the National Academy of Sciences, 97, 7051-7057
In situ hybrididation, 2010, genedetect.com [online] Available at http://www.genedetect.com/insitu.htm.
Soltis DE, Soltis PS, Pires JC, Kovarik A, Tate JA, Mavrodiev E, 2004, Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons, Biological Journal of the Linnean Society. 82, 485-501
Sadava, Purves, Heller, Orians, Hellis, 2008. Life: The science of Biology. 8th Edition, Sinauer Associates.
Cook LM, Soltis PS, 1999, Mating systems of diploid and allotetraploid populations of Tragopogon (Asteraceae). I. Natural Populations, Heredity, 82, 237-244